Jan Coufal

Preferential binding of IFI16 protein to cruciform structure and superhelical DNA.

Interferon (IFN)-inducible HIN-200 proteins play an important role in transcriptional regulation linked to cell cycle control, inflammation, autoimmunity and differentiation. IFI16 has been identified as a target of IFNα and γ and is a member of the HIN-200 protein family. Expression level of IFI16 is often decreased in breast cancers, implicating its role as a tumor suppressor. As a potent transcription factor, IFI16 possesses a transcriptional regulatory region, a PYD/DAPIN/PAAD region which associates with IFN response, DNA-binding domains and binding regions for tumor suppressor proteins BRCA1 and p53. It is also reported that IFI16 protein is capable of binding p53 and cMYC gene promoters. Here, we demonstrate that IFI16 protein binds strongly to negatively superhelical plasmid DNA at a native superhelix density, as evidenced by electrophoretic retardation of supercoiled (sc) DNA in agarose gels. Binding of IFI16 to supercoiled DNA results in the appearance of one or more retarded DNA bands on the gels. After removal of IFI16, the original mobility of the scDNA is recovered. By contrast, IFI16 protein binds very weakly to the same DNA in linear state. Using short oligonucleotide targets, we also detect a strong preference for IFI16 binding to cruciform DNA structure compared to linear DNA topology. Hence, this novel DNA-binding property of IFI16 protein to scDNA and cruciform structures may play critical roles in its tumor suppressor function.

Preferential binding of p53 tumor suppressor to p21 promoter sites that contain inverted repeats capable of forming cruciform structure

p53 Is one of the most critical proteins involved in protecting organisms from malignancies and its gene is frequently mutated in these diseases. p53 Functions as a transcription factor and its role in the cell is mediated by sequence-specific DNA binding. Although the genome contains many p53-binding sequences, the p53 protein binds only a subset of these sequences with high affinity. One likely mechanism of how p53 binds DNA effectively underlies its ability to recognize selective local DNA structure. We analyzed the possibility of cruciform structure formation within different regions of the p21 gene promoter. p53 protein remarkably activates the transcription of p21 gene after genotoxic treatment. In silico analysis showed that p21 gene promoter contains numerous p53 target sequences, some of which have inverted repeats capable of forming cruciform structures. Using chromatin immunoprecipitation, we demonstrated that p53 protein binds preferentially to sequences that not only contain inverted repeats but also have the ability to create local cruciform structures. Gel retardation assay also revealed strong preference of the p53 protein for response element in superhelical state, with cruciform structure in the DNA sequence. Taken together, our results suggest that p53 response elements potential for cruciform structure formation could be an additional determinant in p53 DNA-binding machinery.

Palindrome analyser - A new web-based server for predicting and evaluating inverted repeats in nucleotide sequences.

DNA cruciform structures play an important role in the regulation of natural processes including gene replication and expression, as well as nucleosome structure and recombination. They have also been implicated in the evolution and development of diseases such as cancer and neurodegenerative disorders. Cruciform structures are formed by inverted repeats, and their stability is enhanced by DNA supercoiling and protein binding. They have received broad attention because of their important roles in biology. Computational approaches to study inverted repeats have allowed detailed analysis of genomes. However, currently there are no easily accessible and user-friendly tools that can analyse inverted repeats, especially among long nucleotide sequences. We have developed a web-based server, Palindrome analyser, which is a user-friendly application for analysing inverted repeats in various DNA (or RNA) sequences including genome sequences and oligonucleotides. It allows users to search and retrieve desired gene/nucleotide sequence entries from the NCBI databases, and provides data on length, sequence, locations and energy required for cruciform formation. Palindrome analyser also features an interactive graphical data representation of the distribution of the inverted repeats, with options for sorting according to the length of inverted repeat, length of loop, and number of mismatches. Palindrome analyser can be accessed at http://bioinformatics.ibp.cz.

Recognition of Local DNA Structures by p53 Protein

p53 plays critical roles in regulating cell cycle, apoptosis, senescence and metabolism and is commonly mutated in human cancer. These roles are achieved by interaction with other proteins, but particularly by interaction with DNA. As a transcription factor, p53 is well known to bind consensus target sequences in linear B-DNA. Recent findings indicate that p53 binds with higher affinity to target sequences that form cruciform DNA structure. Moreover, p53 binds very tightly to non-B DNA structures and local DNA structures are increasingly recognized to influence the activity of wild-type and mutant p53. Apart from cruciform structures, p53 binds to quadruplex DNA, triplex DNA, DNA loops, bulged DNA and hemicatenane DNA. In this review, we describe local DNA structures and summarize information about interactions of p53 with these structural DNA motifs. These recent data provide important insights into the complexity of the p53 pathway and the functional consequences of wild-type and mutant p53 activation in normal and tumor cells.

IFI16 Preferentially Binds to DNA with Quadruplex Structure and Enhances DNA Quadruplex Formation

Interferon-inducible protein 16 (IFI16) is a member of the HIN-200 protein family, containing two HIN domains and one PYRIN domain. IFI16 acts as a sensor of viral and bacterial DNA and is important for innate immune responses. IFI16 binds DNA and binding has been described to be DNA length-dependent, but a preference for supercoiled DNA has also been demonstrated. Here we report a specific preference of IFI16 for binding to quadruplex DNA compared to other DNA structures. IFI16 binds to quadruplex DNA with significantly higher affinity than to the same sequence in double stranded DNA. By circular dichroism (CD) spectroscopy we also demonstrated the ability of IFI16 to stabilize quadruplex structures with quadruplex-forming oligonucleotides derived from human telomere (HTEL) sequences and the MYC promotor. A novel H/D exchange mass spectrometry approach was developed to assess protein interactions with quadruplex DNA. Quadruplex DNA changed the IFI16 deuteration profile in parts of the PYRIN domain (aa 0–80) and in structurally identical parts of both HIN domains (aa 271–302 and aa 586–617) compared to single stranded or double stranded DNAs, supporting the preferential affinity of IFI16 for structured DNA. Our results reveal the importance of quadruplex DNA structure in IFI16 binding and improve our understanding of how IFI16 senses DNA. IFI16 selectivity for quadruplex structure provides a mechanistic framework for IFI16 in immunity and cellular processes including DNA damage responses and cell proliferation.

Superhelical DNA as a preferential binding target of 14-3-3γ protein.

The 14-3-3 protein family is a highly conserved and widely distributed group of proteins consisting of multiple isoforms in eukaryotes. Ubiquitously expressed, 14-3-3 proteins play key roles in DNA replication, cell cycle regulation, and apoptosis. The function of 14-3-3 proteins is mediated by interaction with a large number of other proteins and with DNA. It has been demonstrated that 14-3-3γ protein binds strongly to cruciform structures and is crucial for initiating replication. In this study, we analyzed DNA binding properties of the 14-3-3γ isoform to linear and supercoiled DNA. We demonstrate that 14-3-3γ protein binds strongly to long DNA targets, as evidenced by electrophoretic mobility shift assay on agarose gels. Binding of 14-3-3γ to DNA target results in the appearance of blurry, retarded DNA bands. Competition experiments with linear and supercoiled DNA on magnetic beads show very strong preference for supercoiled DNA. We also show by confocal microscopy that 14-3-3 protein in the HCT-116 cell line is co-localized with DNA cruciforms. This implies a role for the 14-3-3γ protein in its binding to local DNA structures which are stabilized by DNA supercoiling.

p73, like its p53 homolog, shows preference for inverted repeats forming cruciforms

p73 is a member of the p53 protein family and has essential functions in several signaling pathways involved in development, differentiation, DNA damage responses and cancer. As a transcription factor, p73 achieves these functions by binding to consensus DNA sequences and p73 shares at least partial target DNA binding sequence specificity with p53. Transcriptional activation by p73 has been demonstrated for more than fifty p53 targets in yeast and/or human cancer cell lines. It has also been shown previously that p53 binding to DNA is strongly dependent on DNA topology and the presence of inverted repeats that can form DNA cruciforms, but whether p73 transcriptional activity has similar dependence has not been investigated. Therefore, we evaluated p73 binding to a set of p53-response elements with identical theoretical binding affinity in their linear state, but different probabilities to form extra helical structures. We show by a yeast-based assay that transactivation in vivo correlated more with the relative propensity of a response element to form cruciforms than to its expected in vitro DNA binding affinity. Structural features of p73 target sites are therefore likely to be an important determinant of its transactivation function.

The Amino Acid Composition of Quadruplex Binding Proteins Reveals a Shared Motif and Predicts New Potential Quadruplex Interactors

The importance of local DNA structures in the regulation of basic cellular processes is an emerging field of research. Amongst local non-B DNA structures, G-quadruplexes are perhaps the most well-characterized to date, and their presence has been demonstrated in many genomes, including that of humans. G-quadruplexes are selectively bound by many regulatory proteins. In this paper, we have analyzed the amino acid composition of all seventy-seven described G-quadruplex binding proteins of Homo sapiens. Our comparison with amino acid frequencies in all human proteins and specific protein subsets (e.g., all nucleic acid binding) revealed unique features of quadruplex binding proteins, with prominent enrichment for glycine (G) and arginine (R). Cluster analysis with bootstrap resampling shows similarities and differences in amino acid composition of particular quadruplex binding proteins. Interestingly, we found that all characterized G-quadruplex binding proteins share a 20 amino acid long motif/domain (RGRGR GRGGG SGGSG GRGRG) which is similar to the previously described RG-rich domain (RRGDG RRRGG GGRGQ GGRGR GGGFKG) of the FRM1 G-quadruplex binding protein. Based on this protein fingerprint, we have predicted a new set of potential G-quadruplex binding proteins sharing this interesting domain rich in glycine and arginine residues.

Correction: Brázda, V. and Coufal, J. Recognition of Local DNA Structures by p53 Protein. Int. J. Mol. Sci. 2017, 18, 375

The authors wish to make the following corrections to their paper [1] [...].